This invention relates to materials and methods for determining the sequence of unknown or target nucleic acids, and more specifically to materials and methods for determining the sequence of target nucleic acids using an electronically-indexed solid phase, with transponders associated with the solid phase particles.
A high throughput method for ascertaining the sequence of sample nucleic acids is sequencing by hybridization (SBH). In that method, a large number of oligonucleotide probes is allowed to interact with the nucleic acid molecules in a sample, and a detection system is provided to determine whether individual oligonucleotides have annealed to the template. Two basic designs have been described. In one, the oligonucleotide probes are arranged in a two-dimensional array on the surface of a membrane, filter, VLSI chip, or the like. In the other, the array on the membrane is formed with a large number of sample DNA sequences, and each membrane is subjected to a series of hybridization steps with different oligonucleotide probes. A label used to monitor the binding, either a radioactive isotope or a fluorophore, is carried on the sample DNA or on the oligonucleotide probe. The sequence is derived from coordinates of spots showing a high level of the label deposition on the two-dimensional arrays.
Conventional SBH methods are limited by difficulties related to preparation of arrays of DNA, non-specific annealing of DNAs, the need for special instrumentation to read the data and the automation of the process and data analysis. In SBH, the sequence is determined by defining the two-dimensional coordinates of relevant dots in the array formed by the deposited DNA molecules. In the present invention, a partial or the complete sequence of the DNA molecule is determined by decoding the electronic memory elements associated with DNA probes of known sequence.
An advantage of the present invention over conventional sequencing methods is that it is extremely fast, because the sequence is deduced from a series of readings of digitally-stored sequences in the transponder, rather than from a series of measurements of a chemical or physical property of DNA, or the location of DNA in an array. The method of this invention is referred to hereinafter as digital sequencing.